(1) Field of the Invention
The invention relates to analyzing organisms such as bacterial spores based on their soluble polypeptides and more particularly to the identification of organisms such as bacterial spores based on peptide fragments of their soluble polypeptides.
(2) Description of Related Art
A number of approaches have been used in the past for applying the analytic power of mass spectrometry to microorganisms (Fenselau and Demirev, Mass Spectrom Rev 20, 157). Among these, electrospray ionization and matrix assisted laser desorption mass spectrometry have provided access to cellular proteins as biomarkers. In many cases proteins have first been isolated from other bacterial material for subsequent analysis by enzymatic, chromatographic and mass spectral procedures (Harris and Reilly, Anal Chem 74, 4410, 2002; Cargile, McLuckey and Stephenson, Anal Chem 73, 1277, 2001; Zhou et al, Proteomics 1, 683, 2001; Krishnamurthy et al, J. Toxicol. Toxin Rev 19, 95, 2000; Xiang et al, Anal Chem 72, 2475, 2000; Arnold and Reilly, Anal Biochem 269, 105, 1999; Holland et al Anal Chem 71, 3226, 1999; Yates and Eng, U.S. Pat. No. 5,538,897; Dai et al, Rapid Commun Mass Spectrom 13, 73, 1999; Liu et al, Anal Chem 70, 1797, 1998; Despeyroux, Phillpotts and Watts, Rapid Commun Mass Spectrom 10, 937, 1996; Cain et al, Rapid Commun. Mass Spectrom 8, 1026, 1994). Isolated proteins were cleaved to peptides, the peptides were partially sequenced by tandem mass spectrometry, and the parent proteins were identified by standard protein and genome database searches. The bacteria species were characterized from the database as the source of the proteins. In other cases researchers have undertaken to analyze protein biomarkers without a separation step (Claydon et al Nature Biotechnol. 14, 1584, 1996; Demirev and Fenselau PCT/US 99/27191; Holland et al, Rapid Commun. Mass Spectrom 10, 1227, 1996; Krishnamurthy, Ross and Rajamani, Rapid Commun. Mass Spetrom 10, 883, 1996; Krishnamurthy U.S. Pat. No. 6,177,266). The sample is lysed on the mass spectrometry sample holder (in situ) and proteins are desorbed directly by matrix assisted laser desorption ionization (MALDI). The spectrum of the mixture of proteins detected can be matched to a carefully prepared library of microbial mass spectra (Conway et al, J. Mol. Microbiol Biotechnol. 3, 103, 2001; Jarman et al, Anal Chem 72, 1217, 2000), allowing distinction of the species, or the microorganism can be characterized by matching the masses of the suite of proteins observed to protein masses predicted from the genome (Demirev et al, Anal Chem 73, 4566, 2001; Pineda et al, Anal Chem 72, 3739, 2000; Demirev et al, Anal Chem 71, 2732, 1999; Demirev and Fenselau PTC/US99/27191).
More recently enzymatic cleavage of proteins on the sample holder for direct analysis of peptides (Yao, Demirev and Fenselau, Anal Chem 74, 2529, 2002; Yao and Fenselau, Rapid Commun Mass Spectrom 16, 1953, 2002) has been proposed to provide a simple rapid analysis of simple viruses. Applying this strategy to more complex microorganisms (Escherichia coli and Erwinia herbicola) has revealed that indiscriminant enzymatic digestion of proteins in a microorganism, without a fractionation step, produces a large mixture of peptides, poor signal to noise ratios, poor sensitivity for tandem mass spectrometry (sequencing) experiments, and poor reproducibility. Also see “Rapid Microorganism Identification by MALDI Mass Spectrometry and Model-derived Ribosomal Protein Biomarkers” Antoine et al., J. Lin, Anal. Chem. 75 (2003) pp 3817-3822; U.S. Pat. No. 6,558,946 to Krishnamurthy and U.S. Pat. No. 6,177,266 to Krishnamurthy et al.; and U.S. Patent Application 20030027231 to Bryden et al.
Among the microorganisms, spores of the genus Bacillus are monitored as important targets in battle spaces, subways and buildings, counter-terrorism activities, and in some medical diagnosis. Direct desorption of biomarker proteins from spores has been challenging, as the outer spore coat is strongly resistant to solvents. Abundant proteins, however, are present within the spore core. These proteins can be extracted from spores by treatment with 1N H; 1 HCl, and hence they are referred in the art to as small, acid-soluble proteins (SASP). Their sequences are different for different spores (Hathout et al, Applied Environ. Microbiology 69, in press, 2003).
Also see WO 02/40678 A1 to Fairhead for a detailed description of small acid-soluble spore proteins, which is hereby incorporated by reference in its entirety.